Cell lines

Active Publication Date: 2015-09-17
MEDIMMUNE LTD
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  • Abstract
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  • Application Information

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Benefits of technology

[0035]As a first aspect of the invention, therefore, there is provided a process for stabilizing a eukaryotic cell line (particularly a mammalian cell line) which expresses PylRS and tRNAPyl and which is suitable for incorporation of a gene encoding a target protein containing one or more non-natural amino acids encoded by an amber codon which comprises culturing said cell line under conditions in which the adverse effect of tRNAPyl expression on cell viability and/or cell growth is reduced or eliminated.
[0036]As a second aspect of the invention, there are provided decoy amino acids (

Problems solved by technology

Thus, without being bound by theory, toxic effects of tRNApyl may be a consequence of imperfect o

Method used

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example 1

Generation of a Stable Cell Line

[0601]The generation of a platform cell line capable of site specific integration of nnAAs into a target protein required the stepwise construction of a cell line stably expressing the pylRS and the pyltRNA. This was accomplished by sequential introduction of the pylRS / tRNA expression elements and iterative selection steps to identify high functioning cells (FIG. 1).

[0602]A plasmid containing nine copies of the U6-pyltRNA expression cassette as well as a sequence encoding the human INF Matrix Attachment Region (pSB-9xtRNA-MARS whereby the U6 is defined in SEQ ID 32, the tRNA sequence in Seq ID28), a sequence element that mediates the organization of chromatin in the nucleus and plays a role in the regulation of gene expression and enhances stability of these elements through replication (Klar 2005; Heng 2004; Piechaczek 1999) were transfected into DG44-CHO cells and selected in ProCHO4 medium supplemented with HT supplement containing 0.1 mM hypoxanth...

example 2

Amber Suppression Associated Toxicity

[0608]During the course of the platform cell line isolation the inventors observed that as an increasing amount of the pylRS / tRNApyl was introduced in order to improve the efficiency of the system, the viability of the cells deteriorated. In particular increasing tRNApyl levels were found to have the greatest impact on amber suppression efficacy. This was observed in cells transiently transfected with pJTI-R4-pylRS-eGFPY40 and vectors encoding different numbers of U6-tRNA expression cassettes and the mean fluorescence was determined using an Accuri flow cytometer (FIG. 4A). We observed that cells lacking a tRNA expression cassette, or cells grown in the absence of ALOC did not show a significant GFP signal. However, the expression level of GFP increased with the number of tRNA gene copies indicating that tRNA is an important component of the amber suppression system. To further refine the effect of tRNA levels in amber suppression we transiently ...

example 3

Techniques to Mitigate Amber Suppression Associated Toxicity

[0613]The toxicity associated with amber suppression led us to conceive of alternative approaches that would mitigate this toxicity in the development of an expression cell line while enabling the isolation of highly active amber suppressor cells.

[0614]“Target First” Approach

[0615]One approach to mitigate the observed toxicity in the development of a stable expression cell line while enabling the isolation of highly active amber suppressor cells requires the introduction of a highly expressed target gene that contains an amber codon prior to the introduction of the pylRS and pyltRNA. High levels of message from this gene effectively compete with endogenenous gene expression for the activated pyltRNA available in the cell and thus reducing the impact to the cell's functional machinery. To do this an eukaryotic expression host cell is transfected with a gene intended for expression and containing one or more amber stop codons...

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Abstract

There is provided inter alia a process for stabilizing a eukaryotic cell line which expresses PylRS and tRNAPyl and which is suitable for incorporation of a gene encoding a target protein containing one or more non-natural amino acids encoded by a nonsense codon which comprises culturing said cell line under conditions in which the adverse effect of tRNAPyl expression on cell viability and/or cell growth is reduced or eliminated.

Description

FIELD OF THE INVENTION[0001]This invention relates to stable eukaryotic cell lines suitable for use in incorporating non natural amino acids into proteins and to processes for preparing them. This invention also relates to proteins with incorporated non natural amino acids which are suitable for conjugation with other proteins, with drugs or other moieties e.g. to allow half life extension and to corresponding protein conjugates. Further, the invention relates to novel amino acid derivatives.BACKGROUND TO THE INVENTION[0002]The site-specific introduction of non natural amino acids (nnAAs) into a target protein provides a significant advantage for the generation of functionalized protein conjugates over non specific methods (Wang et al., 2011). A variety of non natural amino acids are available that contain moieties that provide bioorthogonal sites for conjugation chemistry and enable specific reactions to occur at these sites. Control over the positions of the conjugation site enabl...

Claims

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Application Information

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IPC IPC(8): C12P21/02C07K16/24C07K14/435C12N9/00C07K16/40C12N15/85C07K16/28
CPCC12P21/02C12N15/85C07K16/248C12Y601/01025C12N9/93C07K16/40C07K14/43595C07K16/2863C07C271/12C07C271/20C07C233/49C07C233/56C07C247/04C07K16/244C07K16/32C07K2317/14C07K2317/24C07K2317/31C07K2317/40C07K2317/51C12Y601/01A61K47/6851C07C271/22C12Y301/01025C07C235/74C07K16/46C07K17/08
Inventor GRABSTEIN, KENNETH H.VAN BRUNT, MICHAELMARELLI, MARCELLOBRADY, WILLIAMJOHNSON, JEFFREY
Owner MEDIMMUNE LTD
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